Uncaging

In vitro uncaging at well-defined points

FemtoSmart Galvo equipped with a Multiple beam path extension

The accuracy of the excitation point, and the highly flexible scanning patterns, mean that the FemtoSmart Galvo is the best choice for uncaging experiments. Multiple-point scanning (yellow points on the figure) is used for stimulation around spines, while multiple-line scanning (arrows) makes high-speed imaging along the dendrites possible. The secondary laser beam, essential for the stimulation, is coupled to the existing light path. Thus the stimulation and the imaging can be performed near simultaneously using the galvo scanner. The microsecond-scale switching time between the stimulation and imaging is established by using of Pockels cell.

Specialized software functions for photostimulation mapping

Stimulus mapping and analysis software module

This module of MES control software allows performing photostimulation mapping experiments at a range of locations and shapes. The locations can be picked manually one-by-one, along a line or in a raster, and various stimulation patterns can be selected like point, spiral, x, zigzag. It forms datasets quickly by evaluating fluorescence changes images at defined time intervals. Analysis tool for stimulation mapping can create (multichannel) map images formed by the elicited responses after a mapping experiment.

Compounds for uncaging experiments

DNI-GLU-TFA synthesized by FemtoChemistry

This dinitro-indoline-masked form of glutamate releases the bioactive glutamate more rapidly than any other commercially available compound. It was developed for high-quantum yield requiring less irradiation for release, so its effective concentration is lower than other caging scaffolds. DNI-Glu is a compound developed in-house, only available from Femtonics. See also Chiovini et al., Neuron, 2014.

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